Brightfield images were captured using a Nikon ECLIPSE Ti inverted microscope. Whole-mount immunofluorescence staining was conducted for histological analysis. After fixation, samples were washed three times in DPBS, 5 min each time, followed by 20 min permeabilization in 0.3% Triton-X 100 and 2 h blocking in 1% (w/v) bovine serum albumin (BSA) with 0.1% (v/v) Tween 20. For sarcomeric alpha-actinin (SAA) staining, rabbit anti-SAA antibody (Abcam, cat. ab68167) was applied overnight at 4 °C and anti-rabbit IgG Alexa Fluor 594 (Abcam, cat. ab150080) was subsequently applied at room temperature for 2 h.
Cross-sectional images were acquired for cross-sectional area and specific force assessment. After fixation, EMT samples were dehydrated using 30% (w/v) sucrose solution for 30 min, dried on Kimwipes® (Kimtech Science, cat. 06-666), then embedded in Scigen Tissue-PlusTM OCT compound (Fisher, cat. 23-730-571) and frozen in dry ice overnight. Embedded EMT samples were then cross-sectioned into 4-μm cryosections near the center, permeabilized in 0.3% Triton-X 100 for 3 min, blocked in 1% (w/v) BSA with 0.1% (v/v) Tween 20, then stained for SAA following the above-mentioned procedure. Images were acquired using Leica SP8 inverted confocal microscope and LAS X image processing software.
Cross-sectional images were acquired for cross-sectional area and specific force assessment. After fixation, EMT samples were dehydrated using 30% (w/v) sucrose solution for 30 min, dried on Kimwipes® (Kimtech Science, cat. 06-666), then embedded in Scigen Tissue-PlusTM OCT compound (Fisher, cat. 23-730-571) and frozen in dry ice overnight. Embedded EMT samples were then cross-sectioned into 4-μm cryosections near the center, permeabilized in 0.3% Triton-X 100 for 3 min, blocked in 1% (w/v) BSA with 0.1% (v/v) Tween 20, then stained for SAA following the above-mentioned procedure. Images were acquired using Leica SP8 inverted confocal microscope and LAS X image processing software.